rozwój i odpowiedź na stres

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Udział metabolizmu RNA

w procesach fizjologicznych:

rozwój i odpowiedź na stres

Anna Golisz

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Levels of regulation

I. Chromatin and transcription

II. RNA processing: pre-mRNA splicing

(alternative splicing - AS) and 3’ formation III. RNA stability

IV.Regulation via microRNA

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RNA metabolism regulates most of developmental and signaling processes in plants

► Germination

► Circadian clock

► Transition from vegetative to generative development

► Flowering

► Stress response

Regulation of plant metabolism

I. Chromatin and transcription

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Plant clocks control a plethora of biological processes

2000, vol. 5, no. 12

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The central oscillator

1) CCA1 – CIRCADIAN CLOCK ASSOCIATED 1 LHY – LATE ELONGATED HYPOCOTYL

 MYB transcription factors

 reduction in mRNA levels: negative feedback loop

 mRNA level peaking at dawn

2) TOC1 – TIMING OF CAB EXPRESSION 1

 TOC1 expression oscillates peaking during early evening

(opposite to CCA1 and LHY)

Stratmann & Mas, Seminars in Cell & Developmental Biology (2008) 554–559

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Chromatin-dependent regulation of TOC1

Stratmann & Mas, Seminars in Cell & Developmental Biology 19 (2008) 554–559

HAT – histone acetyltransferase

HDAC – histone deacetylase

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Model for a feedback loop involving LHY, CCA1 and TOC1

3) LHY, CCA1 repress expression of TOC1, their positive regulator

1) PHY and CRY as photoreceptors 2) LHY, CCA1 and TOC1 negative feedback loop

4) Generation of circadian rhythms, including that of CO (CONSTANTS) for flowering time

5) ELF3 (early flowering3) gates the light signals, resetting it at dawn

6) ZTL (ZEITLUPE) and GI (GIGANTEA) also act on light input

FT – FLOWERING LOCUS T

2003, 6:13-19

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Flowering

pathway in plant

Flowering I-II.

Chromatin

and RNA

processing

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Pathways controlling flowering time in A. thaliana

Int. J. Dev. Biol. 2005, 49: 773-780

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Functions of antisense RNAs in the regulation of plant flowering

Rosonina and Manley, Dev. Cell, 2010 Hornyik et al.. RNA UK, 2010

FLC – Flowering Locus C

− MADS box transcription factor

− major repressor of flowering in plants

− expression regulated by FLC antisense

► two major forms of FLC antisense are synthesized

► regulated by alternative polyadenylation by RNA binding proteins, FPA and FCA, and CTSF factor FY

► short asFLC (3’ processing at site I) recruits histone demethylase FLD which introduces transcriptionally repressive histone modifications leading to FLC silencing

► long asFLC (3’ processing at site II) causes nucleosomal rearrangements at

the FLC promoter leading to FLC transcription

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Convergence points in abiotic and biotic stress signaling networks

Fujita et al. Current Opinion in Plant Biology 2006, 9:436–442

I and III. Transcription and RNA stability

Stress response

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Phytohormones – old timers and newcomers

Auxin

Cytokinin Gibberellin

Abscisic Acid

Ethylene

Brassinosteroid Salicylic Acid

Jasmonic Acid

Strigolactone

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Phytohormones regulate all stages of the plant life cycle

Fertilization and fruit formation

Seed

dormancy Embryogenesis

Fruit ripening

Germination

Flower

development

Growth and

branching

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Hormonal responses to abiotic stress

Photooxidative stress

High temperature stress

Water deficit, drought Soil salinity

Air pollution

Wounding and

mechanical damage

Cold and

freezing stress

Plants’ lives are very stressful...

ABA and ethylene help plants respond

to stress

Reprinted by permission from Macmillan Publishers, Ltd. Nature Chemical Biology. Vickers, C.E., Gershenzon, J., Lerdau, M.T., and Loreto, F. (2009) Nature Chemical Biology 5: 283 - 291

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Adapted with permission from RIKEN

Seed quality

Stress tolerance

Dormancy Germination

Biotic stress response

Stomatal aperture Gene expression

Development

Abscisic Acid

controls many plant processes including stress responses, development and reproduction

ABSCISIC ACID (ABA)

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ABA synthesis is strongly induced in response to stress

R.L. Croissant, , Bugwood. www.forestryimages.org . Zabadel, T. J. Plant Physiol. (1974) 53: 125-127.

ABA levels rise during drought stress due in part to increased biosynthesis

Hours of drought stress

Leaf water potential

(atm)

[ABA]

µg/g dry weight

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Abscisic acid (ABA) signaling pathway

Sheard and Zheng NATURE Vol 462/3 December 2009

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There are many genes encoding PYR/PYL/RCARs

Common Name Species Number of genes

Soybean Glycine max 23

Corn Zea mays 20

Western poplar Populus

trichocarpa 14

Rice Oryza sativa 11

Grape Vitis vinifera 8

Sorghum Sorghum bicolor 8

Barrel medic (a model legume)

Medicago

truncatula 6

Arabidopsis Arabidopsis

thaliana 14

Klingler, J.P., Batelli, G., and Zhu, J.-K. J. Exp.Bot. 61: 3199-3210

Raghavendra, A.S., Gonugunta, V.K., Christmann, A., and Grill, E. (2010) Trends Plant Sci. 15: 395-401.

The 14 PYR/RCARs in Arabidopsis

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Schematic model of the transcriptional regulation of ABA signaling by AREB/ABF and ABI5 family TFs

Fujita et al. 2011 J Plant Res

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ahg2-1 – ABA hypersensitive germination

poly(A)-specific ribonuclease AtPARN deadenylation, mRNA degradation

lba1 – ABA-hypersensitive seed germination

RNA helicase UPF1 Nonsense-Mediated decay (NMD) Nonsense-Mediated mRNA decay

sad1 – supersensitive to ABA and drought

LSM complex (Sm-like) snRNP proteins mRNA splicing and degradation

ABA response in RNA metabolic mutants

hyl1 – hypersensitive to salt and ABA RNA binding protein HYL1

miRNA processing and accumulation los4 – sensitive to ABA and cold

putative DEAD box RNA helicase LOS4 mRNA export

m

⁷

G

Lsm1-7

m

⁷

G

PARN

abh1 – hypersensitive response to ABA in germination inhibition nuclear cap-binding protein CBP80

mRNA splicing and stability

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ABA response involves RNA processing and

degradation systems

Hirayama&Shinozaki 2007 TRENDS in Plant Science

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Ethylene (C ₂ H ₄ ) is a gaseous hormone with diverse actions

Ethylene regulates:

 fruit ripening

 organ expansion

 senescence

 gene expression

 stress responses

Cotton plants

7 days ethylene Air (control)

Air Ethylene

Arabidopsis

Beyer, Jr., E.M. (1976) Plant Physiol. 58: 268-271.

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Ethylene responses in Arabidopsis

Lorenzo, O., Piqueras, R., Sanchez-Serrano, J.J., and Solano, R. (2003). Plant Cell 15: 165-178;

Rüžička, K., Ljung, K., Vanneste, S., Podhorská, R., Beeckman, T., Friml, J., and Benková, E. (2007). Plant Cell 19: 2197-2212.

Inhibition of leaf cell expansion

Acceleration of leaf senescence Ethylene-induced gene expression

Inhibition of root

elongation

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=XRN4

Ethylene signal transduction pathway:

XRN4 - 5’-3’ cytoplasmic exoribonuclease

Olmedo et al. PNAS 2006 vol. 103 no. 36

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miRNAs and vegetative phase change

Germination

zygote

JUVENILE PHASE

Vegetative phase change

Vegetative phase change is the transition from juvenile to adult growth in plants

ADULT PHASE

REPRODUCTIVE PHASE EMBRYONIC

PHASE

IV. Regulation via miRNA

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►Small RNAs are a pool of 21 to 24 nt RNAs that generally function in gene silencing

►Small RNAs contribute to post- transcriptional gene silencing by affecting mRNA translation or stability

►Small RNAs contribute to

transcriptional gene silencing through epigenetic modifications to chromatin

AAAAA

RNA Pol Histone modification, DNA methylation

What are small RNAs?

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Leaves are modulated by miRNA activity throughout development

Pulido, A., and Laufs, P. (2010). J.Exp.Bot. 61: 1277-1291

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M.W. Jones-Rhoades et al. Annu. Rev. Plant Biol. 2006. 57:19–53

Phenotypes resulting from microRNA (miRNA) overexpression in Arabidopsis

miRNA156

miRNA164

miRNA172

miRNA319

miRNA166

miRNA159a

miRNA160

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Phase change is specified by miRNAs

HASTY, with a shortened juvenile phase, encodes a protein needed for miRNA

export from nucleus to cytoplasm hasty

Loss-of-function zippy mutants prematurely express adult vegetative traits. ZIPPY

encodes an ARGONAUTE protein, AGO7

Wild-type zippy

Bollman, et al. (2003) Development 130: 1493-1504 Hunter et al. (2003) Curr. Biol. 13: 1734–1739

WT hasty

WT zippy

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Poethig, R.S. (2009) Curr. Opin. Genet. Devel.

miR156 overexpression prolongs juvenile

phase in Arabidopsis

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Reciprocal expression patterns of

MIR156 and MIR172 in the juvenile and adult phase of development

Chuck et al. Current Opinion in Plant Biology 2009, 12:81–86

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miR172 expression temporally regulates AP2-like proteins

It is thought that floral initiation can

occur when the level of AP2-like

floral inhibitors drops below a

certain level

Aukerman, M.J., and Sakai, H. (2003) Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-Like target genes Plant Cell 15: 2730-2741.

WT ^mutant

ap2-12

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Role of conserved plant miRNAs

Khraiwesh et al. 2011 Biochimica et Biophysica Acta

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Regulation of miRNA and their target genes by ABA and salt stress in Arabidopsis

Plant Mol Biol (2009) 71:51–59

The Plant Journal (2007) 49, 592–606

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Khraiwesh et al. 2011 Biochimica et Biophysica Acta

green boxes – upregulated small RNA,

light green boxes – slightly upregulated small RNA red boxes – downregulated small RNA

Stress-regulated small RNAs and their target families

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Regulatory network of stress-responsive miRNAs in Arabidopsis

B. Khraiwesh et al. Biochimica et Biophysica Acta 1819 (2012) 137–148

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Distinctions between animal and plant miRNAs

Animal miRNAs Plant miRNAs

conserved miRNA precursors less conserved mature miRNA pri-miRNA cleaved by DICER

produce 60-70 nt pre-miRNA

pri-miRNA cleaved by Dicer-like1 (DCL1) produce 60-300 nt

miRNA precursor cleaved to miRNA duplex in the

cytoplasm

cleaved to miRNA duplex in the nucleus

miRNA duplex is exported from the nucleus by Exportin-5

miRNA duplex is exported from the nucleus by HASTY (HST) homolog Exportin-5

Lu & Huang BBRC 2008, 368, 458-462

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Wienholds, Plasterk FEBS Letters 579 (2005) 5911–5922

Biological function of miRNAs in animal and disease

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miRNA – control of differentation and development of mammalian cells

Neuronal

differentiation Muscle cell differentation

Cell development

Stefani G., Slack F. J., (2008) Mol Cell Biol

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miRNAs regulate developmental timing

miRNAs were discovered in studies of developmental

progressions in the nematode C. elegans.

A miRNA encoded by lin-4 is required for proper larval

development.

lin-14 gene

3’ untranslated region

lin-4 binding sites

Lee, R.C., Feinbaum, R.L., and Ambrose, V. (1993). Cell 75: 843–845.

Wightman, B., Ha, I., and Ruvkun, G. (1993). Cell 75: 855–862.

lin-14 mRNA lin-4 miRNA

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Downregulation of lin-14 by lin-4 is necessary for normal development

Wild-type C. elegans

lin-4 Loss-of-function lin-4 is a negative

regulator of lin-14 In wild-type worms,

lin-14 is expressed early and then shut off.

lin-14 expression

lin-4 loss-of- function

causes lin-14 expression to remain high.

Lee, R.C., Feinbaum, R.L., and Ambrose, V. (1993). Cell 75: 843–845.

Wightman, B., Ha, I., and Ruvkun, G. (1993). Cell 75: 855–862.

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CurrentOpinioninGenetics&Development 2011, 21:491–497

Regulation of dendritic spine morphogenesis by microRNAs

miR-134 and miR-138 – reduced dendritic spine volume miR-132 – increased dendritic spine density

miR-125 – decreased dendritic spine width and increased length

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► Several miRNAs

(miR183/96/182, miR204, miR211)

are transcriptionally upregulated by light in mouse retinal neurons

► Glutamate transporter SLC1A1 (voltage-dependent) is one of the targets of the light- regulated miRNAs

► miRNAs in retinal neurons decay much faster than in nonneuronal cells

► Blocking action potentials or glutamate receptors

strongly affects miRNA turnover

Light-Regulated Retinal MicroRNAs

Filipowicz et al. Cell 2010, 141, 618-631

microRNA metabolism in neurons is higher than in most other cells types

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miRNA as a DECOY in myeloid cell differentiation

Beitzinger and Meister, Cell, 2010